Anti-CD40L immunotoxins for the treatment of diseases

ABSTRACT

An immunotoxin molecule is described which comprises an antibody specific for human CD40L antigen located on the surface of a human cell, coupled to a toxin molecule or active fragment thereof, wherein the binding of the immunotoxin to the CD40L molecule results in the killing of the CD40L expressing cell. The toxin molecule is especially a type-1 ribosome inactivating protein, or an active fragment thereof. The immunotoxin can be used for the treatment of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, or T-cell malignancies.

This application is a Rule 371 Application of PCT Application No.PCT/NL98/00357 (Publication No. WO 9858678), filed on Jun. 22, 1998which claims priority to EP 97201895.6, filed on Jun. 20, 1997.

FIELD OF THE INVENTION

This invention relates to methods of treating diseases of in which theimmune system is involved. In particular, this invention relates tomethods of treating T-cell mediated autoimmune diseases such as multiplesclerosis and various malignancies of lymphoid origin.

BACKGROUND OF THE INVENTION

Immunotoxins:

Immunotoxins are chimeric molecules in which cell-binding ligands arecoupled to toxins or their subunits. The ligand portion of theimmunotoxin is usually a monoclonal antibody (Mab) that binds toselected target cells. The toxin portion of the immunotoxin can bederived form various sources. Most commonly, toxins are derived fromplants or bacteria, but toxins of human origin or synthetic toxins(drugs) have been used as well. Toxins used for immunotoxins derivedfrom plants or bacteria all inhibit protein synthesis of eukaryoticcells. The most widely used plant toxin ricin, consist of twodisulfate-linked polypeptides A and B (Olsnes et al., in MolecularAction of Toxins and Viruses p51-105 (1982)). The most widely usedbacterial toxin is Pseudomonas exotoxin (PE). Pseudomonas exotoxin isproduced by the bacterium as a single-chain protein (Allured et al.,Proc. Natl. Acad. Sci. USA 83:1320 (1986)). Another group ofplant-derived toxins used in ITs are single-chain proteins (type I RIP),frequently found in plants and have similar enzymatic properties as theA-chain of ricin (reviewed in Stirpe and Barbieri FEBS Lett. 195:1(1986)), these type I RIP however lack the B-chain. The absence ofbinding activity and as a consequence the inability of the native toxinto bind to cells significantly decreases the non-specific toxicity andmakes these toxins extremely interesting for usage in ITs. To targetthese toxins to potential harmful cells, they are coupled to a Mabagainst a specific protein on the surface of these targeted cells. Thecross-linker used to join the Mab and the toxin must remain stableextracellular, but after internalization of the conjugate into the cell,be labile intracellular so that the toxin fragment can be released inthe cytosol and target to the appropriate intracellular location. Acomplete Mab consists of two heavy and two light chains and can bechemically coupled to the toxin. Using this chemical coupling usuallyseveral toxin molecules are coupled to one Mab molecules, resulting inprotein complexes of considerable size. An alternative to complete Mabs,is to use single-chain antibody fragments (scFv), which consist of onlythe variable part of the heavy chain (VH) and the variable part of thelight chain (VL) coupled via a short linker (Pastan et al., Annu. Rev.Biochem. 61:331 (1992)). The usage of scFv-ITs has a number ofadvantages compared to chemically coupled ITs. First, the scFv can becloned via a short linker to a toxin and can be expressed asfusion-protein in a bacterial expression system. Secondly, tissuepenetration is a major obstacle when chemical coupled IT-conjugates wereused in various animal models, the scFv format being superior in thisrespect. The use of a scFv as ligand portion of an immunotoxin reducesthe size of ligand portion with a factor 6 as compared to a complete Maband in these recombinant molecules one toxin molecule is linked to onescFv reducing the size even further. Since a scFv-IT is produced as onemolecule unwanted cleavage of the toxin and the ligand in thecirculation can not occur. The capability of scFv-ITs to specificallyeliminate cells, revealed that intracellular cleavage of the ligand andtoxin part, that is necessary when ITs are used, is not necessary forthe cytotoxic effect of certain scFv-ITs.

Various types of immunotoxins directed against different cellulartargets have been evaluated in vivo, both in animal models and in phaseI or II clinical trials. The vast majority of clinical studies withimmunotoxins has been performed for anti-tumor therapy using ricin Achain or blocked ricin (Frankel et al., Leukemia and Lymphoma 26:28(1997), Lynch et al., J. Clin. Onc. 15:723 (1997)). Reports on theadministration of immunotoxins containing type I RIPs are limited.Thusfar only two studies have been published, the first using saporin-S6coupled to an anti-CD30 Mab (Falini et al., Lancet 339:1195 (1992)) andthe second using the PAP toxin coupled to an anti-CD19 Mab (Uckun etal., Blood 79:3369 (1992)). An increasing number of preclinical studiesusing immunotoxins containing various different type I RIPs (momordin,gelonin, saporin, bryodin and bouganin) are currently under development.

The CD40L Molecule:

The CD40L molecule belongs to the TNF/CD40L gene family (Armitage etal., Curr. Opin. Immunol. 6:407 (1994)). Although TNF is a solublecytokine, it is initially synthesized as a membrane associated molecule.Most of the members of the of the TNF/CD40L receptor family are type IItransmembrane proteins. Initially it was reported that the expression ofthe CD40L was restricted to activated CD4⁺ T cells. Now it has also beendetected on B cells from autoimmuine patients, on mast cells and onbaophils. The cell surface expression of CD40L is tightly regulated,specific signals are needed for its appearance and, once engaged withCD40 the molecule rapidly disappears again.

Autoimmune Diseases:

A normal functioning immune system has self-regulating mechanisms toterminate the immune response when it is no longer needed. When theseself-regulatory mechanisms become compromised, a person may develop aso-called autoimmune disease. Examples of autoimmune diseases arerheumatoid arthritis, multiple sclerosis, type I diabetes, lupus,thyroiditis, systemic lupus erythematosus and myasthenia gravis.

Multiple sclerosis (MS) is a severely disabling progressive neurologicaldisease, involving autoimmune attack against myelin in the centralnervous system. MS affects 1 in 1000 in the USA and Europe. Due toimproved diagnosis that number is currently increasing. Onset of diseaseis usually around 30 years of age and, on average, patients are in needof treatment for another 28 years. Diagnosis of exacerbations and earlyidentification of onset of exacerbations has improved greatly, allowingdesign of novel treatment strategies. Recently, the involvement of theCD40L molecule in the pathophysiology of MS has been demonstrated usingthe experimental allergic encephalomyelitis model in mice (Gerritse etal., Proc. Natl. Acad. Sci. USA 93:2499 (1996)). In this model,injection of mice with a blocking monoclonal antibody (Mab) to CD40L atthe time of disease induction, completely prevents disease. Furthermore,in situ analysis of CD40L and CD40 in human MS brain has revealed thatCD40 expression is abundantly expressed on macrophages in perivascularinfiltrates. Frequencies of CD40L positive cells in these infiltrateswere modest, but could be found in juxtaposition to CD40 positive cells,indicative of an ongoing cellular interaction.

Systemic lupus erythematosus (SLE) is an other autoimmune disease inwhich the CD40L molecule has been implicated. SLE, in contrast to mostautoimmune diseases, has the potential to involve multiple organs. Theclinical manifestations of SLE are extremely variable and diverse. Somepatients only have mild involvement of skin and joints, require littlemedication and show spontaneous remissions. Whereas other patientssuffer from severe and progressive glomerulonephritis that in the enddoes not even respond to high doses steroids and cyclophosphamide. SLEcan manifest at nearly any age, but the disease onset is usually between15 and 50 years. SLE affects about 8 times more females than males. Thechance that a caucasian women in her life time develops SLE is about 1in 700, whereas this incidence can be two to four times higher inhispanics or blacks. The overall prevalence of SLE is in the order 1 in2000. SLE is characterized by a production of high affinity IgGantibodies to self antigens (autoantibodies). The principal targets ofautoantibodies in SLE include certain protein-nucleic acid complexes.The multivalent nature of these complexes and their ability tocross-link B-cell receptors have been proposed as explanations for theirstrong immunogenicity. However, the mechanism by which theseautoantibodies cause disease is still unclear. Autoantibodies tophospholipids are also frequently found and are associated withthrombotic complications. Also autoantibodies to cell surface moleculescan be found. These target specificities are easier to understand withrespect to the pathology, causing problems such as hermolytic anemia andplatelet destruction. In contrast to autoimmune diseases such as RA andMS, T cells do not appear to play a direct role in tissue damage in SLE,although the do play an important role in the production ofautoantibodies. The induction of the CD80/86 molecules onautoantigen-specific B cells by autoantigen-specific helper T cells viathe CD40L-CD40 interaction represents a critical step in the maturationand subsequent differentiation of autoantigen-specific B cells. Recentlyit was observed that both T cells and also B cells from active SLEpatients show a constitutive expression of CD40L (Desai-Metha et al., J.Clin. Invest. 97:2063 (1996)).

SUMMARY OF THE INVENTION

Activated T cells are specifically involved in the pathophysiology ofautoimmune diseases such as MS and SLE. The onset of an exacerbation inautoimmune patients is believed to started when autoreactive T cells areactivated. This antigen-specific activation results in the expresssignificant amounts of CD40L on the cell surface. This has led severalgroups to explore the physical blocking of the CD40L-CD40 interaction asa treatment modality for auto immune diseases. However, it may beexpected that after withdrawal of the therapeutic molecule that blocksthe CD40L-CD40 interaction, the disease can return to the same magnitudeor even more severe as before the treatment. As an alternative, it isbeing explored to selectively inactivate autoantigen-specific T cellswith modified autoantigens. However, it has not previously been proposedto use the CD40L receptor on activated T cells to selectively eliminateautoantigen-specific T cells from circulation of autoimmune patients.The present inventors propose a selective method for the treatment ofautoimmuine diseases such as MS and SLE, that is based on the selectivekilling of the CD40L-positive autoantigen-specific T cells by ananti-CD40L immunotoxin fusion protein. The significant advantage of theuse of an anti-CD40L immunotoxin fusion protein over the use of blockinganti-CD40L Mabs is that after several rounds of anti-CD40L immunotoxintreatment, all autoreactive T cells will have been deleted from thepatients T-cell repertoire possibly resulting in a cure from thedisease.

The current invention is thus based on the discovery that conjugates ofantibodies to human CD40L and a toxin (immunotoxin) can effectively killcells expressing the CD40L molecule. Accordingly, these anti-CD40Limmunotoxins can be used to prevent or treat diseases or conditions thatare mediated by the cells expressing the CD40L molecules. Accordingly,it is a primary object of this invention to provide an immunotoxincomprising a Mab capable of binding to the human CD40L antigen locatedon the surface of activated human lymphocytes and a toxin molecule,wherein the binding of said immunotoxin to the CD40L positive cellresults in cell death.

It is another object of this invention to provide a method for treatingautoimmune diseases such as multiple sclerosis, psoriasis, rheumatoidarthritis and systemic lupus erythematosus in a patient, the methodcomprising administering to a patient in need of such treatment atherapeutically effective amount of an immunotoxin capable of binding tothe human CD40L antigen located an the surface of activated humanlymphocytes, wherein the binding of said immunotoxin to the CD40Lantigen results in elimination of the CD40L expressing cells andinhibition of the local inflammatory response, in a pharmaceuticallyacceptable excipient.

It is a further object of this invention to provide a method fortreating malignancies of lymphoid origin in a patient, the methodcomprising administering to a patient in need of such treatment atherapeutically effective amount of an IT capable of binding to thehuman CD40L antigen located on the surface of malignant cells oflymphoid origin, wherein the binding of the IT to the CD40L antigenresults in elimination of the CD40L expressing tumor cells, in apharmaceutically acceptable excipient.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All of thesepublications and applications, cited previously or below are herebyincorporated by reference.

As used herein, the term “immunotoxin” refers to chimeric molecules inwhich a cell binding ligand is coupled a toxin or its sub-unit. Thetoxin portion of the immunotoxin can be derived from various sources,such as plants or bacteria, but toxins of human origin or synthetictoxins (drugs) can be used as well.

Preferably, the toxin part is derived form a plant toxin, such as aribosome inactivating protein (RIP), type-1 or type-2. A type-2 RIPincludes for example ricin (see Olsnes et al, 1982, above). Type-1 RIP'sare particularly useful for constructing immunotoxins according to theinvention. Type-1 RIP's include pokeweed anti-viral protein (PAP)(reviewed by Irvin, in Antiviral Proteins in Higher Plants 65 (1994)),bryodin (see e.g. EP-A-710723, U.S. Pat. No. 5,597,569 and EP-A-725823,GB-A-2194948), momordin (see WO97/19957, WO92/14491, JP-A-51-67714),gelonin (see e.g. WO94/26910, WO93/09130, WO93/20848, WO93/05168),saporin (see Falini et al, 1992, above), and, bouganin (see co-pendingpatent application PCT/NL98/00336). An example of bacterial toxins isPseudomonas exotoxin (see EP-A-583794, Allured et al, 1986 above).

The term “ligand” may refer to all molecules capable of binding with orotherwise recognizing a receptor on a target cell. Examples of suchligands include, but are not limited to, antibodies, growth factors,cytokines, hormones and the like, that specifically bind desired targetcells.

As used herein, the term “antibody” refers to polyclonal antibodies,monoclonal antibodies, humanized antibodies, single-chain antibodies,and fragments thereof such as Fab, F(ab′)2, Fv, and other fragmentswhich retain the antigen binding function of the parent antibody.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and others which retain the antigen binding function of theantibody. Monoclonal antibodies of any mammalian species can be used inthis invention. In practice, however, the antibodies will typically beof rat or murine origin because of the availability of rat or murinecell lines for use in making the required hybrid cell lines orhybridomas to produce monoclonal antibodies.

As used herein, the term “humanized antibodies” means that at least aportion of the framework regions of an immunoglobulin are derived fromhuman immunoglobulin sequences.

As used herein, the term “single chain antibodies” refer to antibodiesprepared by determining the binding domains (both heavy and lightchains) of a binding antibody, and supplying a linking moiety whichpermits preservation of the binding function. This forms, in essence, aradically abbreviated antibody, having only that part of the variabledomain necessary for binding to the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al., Methods for the generation of antibodiessuitable for use in the present invention are well known to thoseskilled in the art and can be found described in such publications asHarlow & Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, (1988).

The immunotoxin molecules of the present invention may be generated byconjugation of a CD40L binding ligand to a toxin by any method known andavailable to those skilled in the art. The ligand and the toxinmolecules may be chemically bonded together by any of a variety ofwell-known chemical procedures, such as the use of heterobifunctionalcross-linkers, e.g. SPDP, carbodiimide or glutaraldehyde. Production ofvarious immunotoxins is well-known within the art and can be found, forexample in “Monoclonal Antibody-Toxin Conjugates: Aiming the MagicBullet”, Thorpe et al., Monoclonal Antibodies in Clinical Medicine,Academic Press, pp.168-190 (1982) and Waldmann, Science, 252:1657(1991), both of which are incorporated by reference.

The ligand may also be fused to the toxin by recombinant means such asthrough the production of single chain antibody-toxin fusion proteins.The genes encoding ligand and the toxin may be cloned in cDNA form andlinked directly or separated by a small peptide linker by any cloningprocedure known to those skilled in the art. See for example Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harborlaboratory, (1989).

A person skilled in the art will realize that additional modifications,deletions and insertions may be made to the ligand binding agent and thetoxin genes. Especially, deletions or changes may be made in the linkerconnecting a ligand gene to the toxin. All such constructions may bemade by methods of genetic engineering well known to those skilled inthe art (see, generally, Sambrook et al., supra) and may produceproteins that have differing properties of affinity, specificity,stability and toxicity that make them particularly suitable for variousclinical or biological applications.

The pharmaceutical compositions of this invention are administered at aconcentration that is therapeutically effective to a patient in the needof a treatment. To accomplish this goal, the pharmaceutical compositionsmay be formulated using a variety of acceptable excipients known in theart. The compositions for administration will commonly comprise asolution of the anti-CD40L immunotoxin molecule dissolved in apharmaceutical acceptable carrier, preferably an aqueous carrier.Typically, the immunotoxins are administered by injection, eitherintravenously, intraperitoneally, in an other body cavity or or into alumen of an organ. Methods to accomplish this administration are knownto those of ordinary skill in the art. It may also be possible to obtaincompositions which may be topically or orally administered, or which maybe capable of transmission across mucous membranes.

Before administration to patients, formulants may be added to theantibodies. A liquid formulation is preferred. For example, theseformulants may include oils, polymers, vitamins, carbohydrates, aminoacids, salts, buffers, albumin, surfactants, or bulking agents.Preferably carbohydrates include sugar or sugar alcohols such as mono-,di-, or polysaccharides. The saccharides can include fructose, glucose,mannose, sorbose, xylose, lactose, maltose, sucrose, dextan, pullulan,dextrin, α- and β-cyclodextrin, soluble starch, hydroxyethyl starch,carboxymethyl cellulose, other water-soluble glucans, or mixturesthereof. Sucrose is most preferred. “Sugar alcohol” is defined as a C₄to C₈ hydrocarbon having an —OH group and includes galactitol, inositol,mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is mostpreferred. These sugars or sugar alcohols mentioned above may be usedindividually or in combination. There is no fixed limit to amount usedas long as the sugar or sugar alcohol is soluble in the aqueouspreparation. Preferably, the sugar or sugar alcohol concentration isbetween 1.0 w/v % and 7.0 w/v %, more preferable between 2.0 and 6.0 w/v%. Preferably amino acids include levorotary (L) forms of carnitine,arginine, and betaine; however, other amino acids may be added.Preferred polymers include polyvinylpyrrolidone (PVP) with an averagemolecular weight between 2,000 and 3,000, or polyethylene glycol (PEG)with an average molecular weight between 3,000 and 5,000. It is alsopreferred to use a buffer in the composition to minimize pH changes inthe solution before lyophilization or after reconstitution. Most anyphysiological buffer may be used, but citrate, phosphate, succinate, andglutamate buffers or mixtures thereof are preferred. Most preferred is acitrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar.Surfactants that can be added to the formulation are shown in EP Nos.270,799 and 268,110.

Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. Preferably, the protectivegroup has between 1 and 8 carbons, more preferably it is methyl. Thesymbol n is a positive integer, preferably between 1 and 1,000, morepreferably between 2 and 500. The PEG has a preferred average molecularweight between 1000 and 40,000, more preferably between 2000 and 20,000,most preferably between 3,000 and 12,000. Preferably, PEG has at leastone hydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/antibody of the present invention.

Water soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), etc. POG is preferred. Onereason is because the glycerol backbone of polyoxyethylated glycerol isthe same backbone occurring naturally in, for example, animals andhumans in mono-, di-, triglycerides. Therefore, this branching would notnecessarily be seen as a foreign agent in the body. The POG has apreferred molecular weight in the same range as PEG. The structure forPOG is shown in Knauf et al., 1988, J. Biol. Chem. 263:15064, and adiscussion of POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106,both of which are hereby incorporated by reference in their entireties.

Another drug delivery system for increasing circulatory half-life is theliposome. Methods of preparing liposome delivery systems are discussedin Gabizon et al., Cancer Research (1982) 42:4734; Cafiso, BiochemBiophys Acta (1981) 649:129; and Szoka, Ann Rev Biophys Eng (1980)9:467. Other drug delivery systems are known in the art and aredescribed in, e.g., Poznansky et al., Drug delivery systems (R. L.Juliano, ed., Oxford, N.Y. 1980), pp. 253-315; M. L. Poznansky, PharmRevs (1984) 36:277.

After the liquid pharmaceutical composition is prepared, it ispreferably lyophilized to prevent degradation and to preserve sterility.Methods for lyophilizing liquid compositions are known to those ofordinary skill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is preferablyadministered to subjects using those methods that are known to thoseskilled in the art.

As stated above, the immunotoxins and compositions of this invention areused to treat human patients. The preferred route of administration isparenterally. In parenteral administration, the compositions of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with a pharmaceuticallyacceptable parenteral vehicle. Such vehicles are inherently nontoxic andnontherapeutic. Examples of such vehicles are saline, Ringer's solution,dextrose solution, and Hanks+ solution. Nonaqueous vehicles such asfixed oils and ethyl oleate may also be used. A preferred vehicle is 5%dextrose in saline. The vehicle may contain minor amounts of additivessuch as substances that enhance isotonicity and chemical stability,including buffers and preservatives.

The dosage and mode of administration will depend on the individual.Generally, the compositions are administered so that antibodies aregiven at a dose between 1 :g/kg and 20 mg/kg, more preferably between 20:g/kg and 10 mg/kg. Preferably, it is given as a bolus dose. Continuousinfusion may also be used, if so, the antibodies may be infused at adose between 1 and 100 :g/kg/min.

The compositions containing the present pharmaceutical compositions or acocktail thereof (i.e., with other pharmaceutically active proteins) canbe administered for therapeutic treatments. In therapeutic applications,compositions are administered to a patient suffering from a disease, inan amount sufficient to cure or at least partially arrest the diseaseand its complications. An amount adequate to accomplish this is definedas a “therapeutically effective dose”. Amounts effective for this usewill depend upon the severity of the disease and the general state ofthe patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES Materials and Methods

Cell Lines and Culture Conditions

The B-cell line JY was cultured in T75 culture flasks routinely (Costar,Cambridge, Mass., USA) in Iscove's modified Dulbecco's medium (IMDM) towhich 50 mg/ml gentamycin and 10% foetal calf serum was added (FCS)(Hyclone, Logan, Utah USA). The cells were cultured in a humidifiedincubator at 37° C. and 5% CO₂. Every week the cells were split (1/20 to1/100). To store the cell line, ampoules were made containing 5-10×10⁶cells/ml Hank's balanced salt solution HBSS supplemented with 20% FCSand 10% DMSO and stored in the liquid nitrogen. A sub-clone of theJurkatt T-cell line (SPVJ-39.8) constitutively expressing CD40L wascultured under similar condition using glutamine-supplemented RPMI 1640as culture medium.

Lymphocyte Isolation and Stimulation

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedblood from healthy donors by Ficoll-Hypaque density centrifugation andresuspended in complete medium consisting of RPMI 1640 (Gibco, Paisley,UK) supplemented with 2 mM L-glutamine, streptomycin (100 mg/ml),penicillin (100 U/ml) and 5% heat-inactivated autologous plasma. T cellswere further purified from the PBMC preparations by depletion ofmonocytes, B cells and NK cells using Lumpho-Kwik T (One Lambda, LosAngeles, Calif., USA) according to the manufacturers protocol. Forinduction of CD40L on T cells, PBMC or purified T-cells were stimulatedfor 2-6 hours with PMA and ionomycin.

Polymerase Chain Reaction

To amplify DNA fragments, polymerase chain reactions (PCR) wereperformed. A typical PCR reactions mix contained: 0-10 mM MgCl₂, 50 mMKCl, 10 mM Tris-HCl pH 9.0, 1.0% Triton X-100, 0.25 mM dNTP each, 25pmol primer/100 μl reactions mix, 1-1000 ng DNA/100 μl reaction mix and2.5 U Taq polymerase. Reactions were run using a Perkin Elmerthermocycler (Perkin Elmer Corp, Norwalk Conn.). A standard PCR schemeconsisted of one step for 2-5 min. at 95° C. to denaturate the DNA,followed by 20-40 cycles of 1 min. at 95° C., 1 min. at 55° C. and 1-4min. at 72° C. After the final step an extension step was performed for7 min. at 72° C.

Flow Cytometric Analysis

Cells (0.1-0.2×10⁶/sample) were incubated for 20 min. at 4° C. with thespecific Mab (0.1-1 mg/sample). After washing with FACS buffer (PBSpH7.4 1% BSA 0.1% NaN₃), the cells were incubated for another 20 min. at4° C. with goat anti-mouse Mabs conjugated to fluorescein isothiocyanate(FITC) or phycoerythin (PE). The cells were washed with FACS buffer andfinally suspended in FACS buffer containing 0.5% paraformaldehyde andanalyzed with a FACS can flow cytometer (Becton Dickinson). The specificbinding of the monoclonal antibodies is expressed as the meanfluorescent intensity in arbitrary units. A similar protocol was used totest the single chain antibody expressing phage particles. In this casedetection was done by using an un-conjugated sheep anti-M13 antibody(Pharmacia Uppsala Sweden), followed after washing by incubation withdonkey anti-sheep conjugated to FITC (Sigma Chemical Co. St. Louis, Mo.,USA).

Protein Synthesis Inhibition Assays

The cytotoxic effect of the immunotoxins on cells was assessed bymeasuring their ability to inhibit protein synthesis in aconcentration-dependent way. Cells were seeded in a 96-well round bottomplate and incubated with specific Mab alone, Mab and saporin-labeledgoat anti-mouse immunoglobulins (GAM-sap), GAM-sap alone, anti-CD40L Mabconjugated to saporin (CD40L-sap) or control Mab conjugated to saporin(Con-sap), for various time intervals. Hereafter [³H]-leucine (1 μCi)was added to each well followed by an overnight incubation. Cells wereharvested on glass-wool filters and counted on a beta plate scanner.Cell numbers used were chosen so, that [³H]-leucine incorporation was alinear function of the number of cells. Results were expressed aspercentage ³H-leucine incorporation with regard to mock-treated cells.The IC₅₀ value is the concentration of immunotoxin needed to obtain 50%inhibition of leucine incorporation.

Ribosome inactivation activity of free and conjugated toxins was testedin a reticulocyte lysate system as described by Parente et al. (BiochemBiophys Acta 1216:43 (1993)). Reaction mixtures contained, in a finalvolume of 62.5 μl: 10 mM Tris/HCl buffer, pH 7.4, 100 mM ammoniumacetate, 2 mM magnesium acetate, 1 mM ATP, 0.2 mM GTP, 15 mMphosphocreatine, 3 μg of creatine kinase, 0.05 mM amino acids (minusleucine), 89 nCi of L-[¹⁴C]-leucine, and 25 μl of rabbit reticulocytelysate. Incubation was at 28° C. for 5 min.

Toxicity to Hematopoietic Progenitor Cells (HPC)

Bone marrow mononuclear cells were resuspended in RPMI 1640 containing10% AB serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/mlstreptomycin with or without 10⁻⁸ M, anti-CD40L Mab conjugated tosaporin or anti-CD40L Mab and saporin separately. For the enumeration ofcolony-forming unit-granulocyte/-macrophage (CFU-GM) colonies 100 U/mlGM-CSF and 10 U/ml IL-3 were added, for burst-formingunit-erythroid/macrophage/megakaryocyte (CFU-GEMM) 10 U/ml IL-3 and 3U/ml Epo. Methylcellulose was added to a final concentration of 0.9%.Finally the cells (200,000) were plated out in 3 cm petri dishes andincubated at 37° C. and 5% CO₂. After 14 days colonies of >20 cells werecounted.

Example 1 Generation of Chemically Coupled Anti-CD40L Immunotoxins

Anti-CD40L immunotoxins were prepared essentially according to themethod described by Tazzari et al. (Br. J. Haematol. 81:203 (1992)) andconsisted of Mab conjugated to the type-I ribosome-inactivating proteinbouganin. The Mab and bouganin were conjugated via a disulfide bondbetween added sulfhydryl (SH) groups. Briefly, SH groups were introducedseparately in the Mabs and in bouganin by 2-iminothiolane treatment. Toobtain an optimal bouganin/Mab ratio, the experimental conditions werechosen so, that per bouganin or Mab molecule respectively 1 and 2 SHgroups were introduced (respectively 1 and 0.6 mM 2-iminothiolane wasadded in 50 mM sodium-borate buffer, pH 9). To quantify the amount ofbouganin conjugated in the resulting immunotoxin, a trace of¹²⁵I-labelled bouganin was added to the solution. Ellman's reagent wasadded to determine the number of introduced SH groups and to protect theSH groups to avoid self conjugation of bouganin or Mab. The excess ofEllman's reagent was removed by Sephadex G-25 gel filtration. Themodified bouganin was reduced with 20 mM β-mercapto-ethanol to free itsSH groups and separated from β-mercapto-ethanol by chromatography on aSephadex G-25 column and was collected directly onto the unreducedderivatized Mab. After concentration, the conjugation was allowed toproceed for 16 hours at room temperature. The immunotoxins werecollected from this reaction mixture by gel filtration on SephacrylS-200. Conjugation and all gel filtrations were performed in phosphatebuffered saline, pH 7.5. The Mab and bouganin content of theimmunotoxins was estimated by the absorbance at A₂₈₀ and from the amountof radioactivity.

Example 2 Cloning of Anti-CD40L Monoclonal Antibodies

For the generation of anti-CD40L immunotoxins first the variable heavy(VH) and light chain (VL) of anti-CD40L Mabs are cloned and sequenced.Messenger RNA is prepared from each hybridoma cell line producinganti-CD40L Mabs. Briefly, the cells are lysed after washing in 5 Mguanidinium thiocyanate, followed by an incubation with oligo-dT beadsin order to bind the mRNA. After elution the mRNA is used in a RT-PCRwith degenerated primers either directed against IgG variable lightchain or heavy chain to amplify the variable regions. The PCR productsare cloned for sequence analysis in a plasmid (PcDNA or pUC18). AfterDNA sequencing of various clones the consensus heavy and light chainamino acid sequence of anti-CD40L Mabs are obtained.

Example 3 Generation of Recombinant Anti-CD40L Immunotoxins

To obtain a single chain immunotoxin based on anti-CD40L Mab andbouganin, we use a strategy by which a single chain antibody fragment(scFv) is transferred to an expression cassette system containing thepelB leader signal, the cDNA encoding bouganin and a 6× his purificationtag. In this expression plasmid, the scFv is cloned between the pelBleader signal and a cDNA encoding the type-I RIP bouganin (Bolognesi etal., Planta 203:422 (1997)). In this expression plasmid, the scFv's arecloned between the pelB leader signal and bouganin.

The CD40L-bouganin plasmid contains the Lac promoter that allows theexpression of the immunotoxins after IPTG(isopropyl-8-D-thiogalactopyranoside) induction. BL21DE3 bacteria aretransformed by the CaCl₂ method with the expression plasmid and platedon LB plates containing 100 μg/ml ampicillin. One colony is picked andgrow overnight in LB containing 100 μg/ml ampicillin. Next day theculture is diluted (1/100) in LB containing 100 μg/ml ampicillin untilthe OD₆₀₀ reaches ˜0.5. At this point IPTG (Sigma Chemical Co. St.Louis, Mo., USA)(0.1-1 mM) is added. After 3 hours the cells areharvested for purification of the recombinant scFv-immunotoxin. Topurify the proteins from the periplasmic space, first the cells areharvested by centrifugation at 4000×g for 20 min. and resuspended in 30mM Tris/HCl, 20% sucrose, 0.5 mM EDTA, pH 8.0 and incubated on ice for10 min. Subsequently the cells are centrifugated at 8000×g for 20 min.and resuspended in ice cold 5 mM MgSO₄ followed by incubation on ice for10 min. After centrifugation at 8000×g the supernatant, which containsproteins from the periplasmic space, is collected and dialyzed against50 mM Na-phosphate, 300 mM NaCl, pH 8.0. This preparation is loaded on aNi-NTA column (Qiagen, Chatsworth, USA), subsequently the column iswashed with 50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 6.0 andelution of the recombinant immunotoxins is done by 50 mM Na-phosphate,300 mM NaCl, 10% glycerol, pH 4.0. Column fractions are analyzed onSDS-PAGE; fractions containing immunotoxins are pooled and dialyzedagainst suitable buffer.

The above protocol is intended for use when the immunotoxins aresecreted as soluble proteins in the periplasmic space of the bacteria.In case the immunotoxins appear to be insoluble, a similar purificationprotocol is used based on solubilization of the E.coli cells in 6 Mguandine hypochloride pH 8.0 followed column steps using 8 M Urea pH 8.0and pH 6.0 to remove non-specific binding and elation with 8 M Urea pH4.0. Column fractions containing the recombinant immunotoxins arestepwise dialyzed against a suitable buffer to renatured the proteins.

Example 4 Expression of CD40L on Cell Lines and Toxic Activity ofAnti-CD40L Immunotoxins to Cells and Cell Lines

To test the activity and specificity of the anti-human CD40Limmunotoxins, two cell lines were selected. The first cell line, theJurkat clone SPVJ-39.8 expresses CD40L on the cell surface. As controlwe used the CD40L negative JY B-cell line. The cells are cultured asdescribed in the materials and methods section above and analyzed forexpression of several cell surface markers. In agreement with the cellsurface expression, the anti-human CD40L IT is able to kill theSPVJ-39.8 cells, but not the CD40L-negative JY cells. Additionalexperiments were performed with an anti-mouse CD40L immunotoxin. Theanti-mouse CD40L Mab MR1 was coupled to bouganin as described in thematerials and methods section above. The resulting IT was tested for itscapacity to inhibit the activation of T cells after stimulation withionomycin and phorbol-12-myristate 13-acetate (PMA) to induce CD40L onthe cells. T cells for this experiment were enriched from splenocytes ofBalb/c mice by passing the splenocytes through a nylon wool column. Thismethod yields between 92-95% of CD3⁺ T cells. Collected cells wereresuspended in RPMI with 10% fetal calf serum (FCS) and 100 μM2-mercaptoethanol. Subsequently, 5×10⁶ cells/ml were then cultured inthe presence of mitogenic concentrations of PEA and ionomycin (10 ng/mland 1 μg/ml respectively) in 96-wells microtiter plates. After 3 hoursof incubation at 37° C. in 5% CO₂, titrated concentrations ofunconjugated anti-CD40L Mab or bouganin-conjugated anti-CD40L Mab wereadded to the cultures. 3[H]-thymidine was added during the last 8 hoursof a 3 day-culture. Incorporation of 3[H]-thymidine into proliferatingcells was measured in a beta counter and expressed as counts per min.(cpm) of triplicate cultures. CD40L expression on the isolated T-cellswas upregulated after stimulation of PMA and ionomycin, however asubpopulation of the cells remains negative for CD40L after stimulation(FIG. 1). In FIG. 2, it can clearly be seen that the bouganin-conjugatedanti-CD40L Mab in contrast to unconjugated anti-CD40L Mab has a potentinhibitory effect on the PMA and ionomycin stimulated T-cells.

Example 5 Therapeutic Activity of Anti-CD40L Immunotoxin in a MurineModel for Human Multiple Sclerosis

Experimental allergic encephalomyelitis (EAE) is a murine model forhuman autoimmune disease multiple sclerosis. EAE was induced in inbredfemale SJL/J mice (9-11 weeks old, Harlan CPB, Zeist, The Netherlands)using PLP 139-151 (HSLGKWLGH PDKF (SEQ. ID. NO.2), PeptidesInternational, Louisville, Ky.). On day 0, the mice were injected ineach of their hind foot pads with 50 μl of an emulsion consisting of anequal volume mixture of PLP in phosphate-buffered saline (PBS) andMycobacterium tuberculosis H37-Ra (4 mg/ml, Difco, Detroit, Mich.) incomplete Freund's adjuvant (CFA). Pertussis toxin (Sigma, St. Louis,Mo.), 230 ng in 50 μl of PBS, was administered intravenously, at thesame time and again 2 days later. Mice were injected intraperitoneallywith either unconjugated anti-CD40L Mab at concentrations of 12, 25, 50or 125 μg in 200 μl of PBS or bouganin-conjugated anti-CD40L Mab atconcentrations of 12, 25 or 50 μg on days 0, 2 and 4. EAE signs werescored daily according to the following scale: 0, noclinical disease; 1,flaccid tail; 2, single hind leg paralysis; 3, dual hind leg paralysis;4, moribund; 5, death. FIG. 3 clearly demonstrates that thebouganin-conjugated anti-mouse CD40L Mab has a potent therapeutic effectat the lowest concentration used 12 μg), since treatment results in amilder and much shorter disease period.

2 1 149 PRT Bougainvillea spectabilis 1 Tyr Asn Thr Val Ser Phe Asn LeuGly Glu Ala Tyr Glu Tyr Pro Thr 1 5 10 15 Phe Ile Gln Asp Leu Arg AsnGlu Leu Ala Lys Gly Thr Pro Val Cys 20 25 30 Gln Leu Pro Val Thr Leu GlnThr Ile Ala Asp Asp Lys Arg Phe Val 35 40 45 Leu Val Asp Ile Thr Thr ThrSer Lys Lys Thr Val Lys Val Ala Ile 50 55 60 Asp Val Thr Asp Val Tyr ValVal Gly Tyr Gln Asp Lys Trp Asp Gly 65 70 75 80 Lys Asp Arg Ala Val PheLeu Asp Lys Val Pro Thr Val Ala Thr Ser 85 90 95 Lys Leu Phe Pro Gly ValThr Asn Arg Val Thr Leu Thr Phe Asp Gly 100 105 110 Ser Tyr Gln Lys LeuVal Asn Ala Ala Lys Val Asp Arg Lys Asp Leu 115 120 125 Glu Leu Gly ValTyr Lys Leu Glu Phe Ser Ile Glu Ala Ile Trp Gly 130 135 140 Lys Thr GlnAsn Gly 145 2 13 PRT Homo sapiens 2 His Ser Leu Gly Lys Trp Leu Gly HisPro Asp Lys Phe 1 5 10

What is claimed is:
 1. An immunotoxin molecule comprising an antibody,or a binding fragment thereof, specific for human CD40L antigenconjugated to a Type I ribosome inactivating protein comprising SEQ IDNO 1, wherein the binding of said immunotoxin molecule to the CD40Lmolecule results in the killing of the CD40L expressing cell.
 2. Theimmunotoxin molecule of claim 1, wherein the antibody is monoclonal,chimeric, human, humanized, bispecific, or a heteroconjugate.
 3. Theimmunotoxin molecule of claim 1, wherein the antibody binding fragmentis F(ab′)₂, F(ab)₂, Fab′, Fab.
 4. A composition comprising theimmunotoxin according to claim 1 and a physiologically acceptablediluent, carrier, and/or excipient.
 5. The immunotoxin molecule of claim1, wherein the antibody or binding fragment thereof, and the toxinmolecule are encoded by a single recombinant DNA molecule.
 6. Theimmunotoxin molecule of claim 1, wherein the antibody and toxin moleculeare expressed in a suitable host cell from a recombinant immunotoxinmolecule.
 7. An immunotoxin molecule comprising an antibody, or abinding fragment thereof, specific for human CD40L antigen conjugated toa Type I ribosome inactivating protein isolated from Bougainvilleaspectabilis Willd characterized as having a molecular weight of about26,000 daltons by polyacrylamide gel electrophoresis under reducing andnon-reducing conditions, wherein the binding of said immunotoxinmolecule to the CD40L molecule results in the killing of the CD40Lexpressing cell.
 8. The immunotoxin molecule of claim 7, wherein theantibody is monoclonal, chimeric, human, humanized, bispecific, or aheteroconjugate.
 9. The immunotoxin molecule of claim 7, wherein theantibody binding fragment is F(ab′)₂, F(ab)₂, Fab′, Fab.
 10. Acomposition comprising the immunotoxin according to claim 7 and aphysiologically acceptable diluent, carrier, and/or excipient.
 11. Theimmunotoxin molecule of claim 7, wherein the antibody and toxin moleculeare expressed in a suitable host cell from a recombinant immunotoxinmolecule.